The structure-function relationship and the enzymology involved in the intracellular processing of cathepsins and other hydrolases will be examined. These studies will permit a greater insight into the regulation and pathology involving intracellular hydrolases. The importance of this understanding is underscored by the existence of over 30 diseases related to the disorders of lysosomal enzymes. All intracellular hydrolases so far studied are synthesized as larger proenzymes and proteolytically processed to multichain mature enzymes. The proteolytic process may involve in several steps. The enzymes involved in the proteolytic processing of prohydrolases and their effects on the intracellular hydrolase activities are not known. This project proposes to use the best known cathepsins as models to examine their processing from procathepsins as well as their roles in the processing of other prohydrolases. The specific areas to be addressed are as follows: (1) The structures of proenzymes will be determined by cloning and sequencing their cDNA's. The processing intermediates will be studied by pulse-chase experiments. The processing points will be determined by the N-terminal sequences of the intermediates. (2) The hypothesis of an activation cascade involving the activation of prohydrases will be tested. This will be accomplished by developing an in vitro model for prohydrolase activation and using specific protease inhibitors to examine their roles. The activities of the proteases involved in individual processing steps will be studied using purified enzymes. (3) Glycosidases involved in the unique processing of cathepsin B carbohydrate to a single N-acetylglucosamine will be studied. A possible structural marker on cathepsin B which is recognized for carbohydrate processing will be examined.